Nuclear packing efficiency

ABSTRACT

Methods and devices for determining the nuclear packing efficiency (NPE) of a cell nucleus and other biological particles. An NPE can be determined by correlating at least one biochemical component, such as DNA content, to nuclear volume using a variety of mathematical techniques. Flow cytometry is particularly useful for measuring nuclear volume in terms of the electronic nuclear volume (ENV). The NPE can then be used to characterize individual cells and cell populations in terms of species and tissue source, sexing, stage of the cell division cycle, differentiation and apoptosis, as well as differentiating among benign, malignant and metastatic states to diagnose cancer.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to cell biology, and moreparticularly to characterizing a cell by its nuclear packing efficiency(NPE).

[0003] 2. Background Information

[0004] Virtually all eucaryotic cells have a nucleus, a compartmentenclosed by the nuclear membrane and containing most of the cell's DNA,as well as other nuclear components. Aberrations in the size and shapeof the nucleus have long been recognized as an indication of cancer andother diseases, although their characterization was previously limitedto microscopic observation.

[0005] Since then, attempts have been made to characterize cells byquantitating and comparing various nuclear components. For example,measurements of DNA, RNA and nuclear protein have been compared witheach other. One study measured the quantity of DNA and tried tocorrelate it with measurements of the size of the nucleus, as measuredby light scatter, time of flight and area. However, these correlationshave been hampered by unreliable indirect estimates of the nuclearvolume.

[0006] Indirect estimates of volume from diameter measurements usinglight scatter have worked best assuming uniform spherical particles of acertain size range and having a relatively high index of refraction. Butthis technique becomes less accurate when applied to nonideal biologicalsamples outside the optimal range of measurement. Other measurements,termed “time-of-flight” or TOF, measure the size of particles as aflowstream carries the particles across a beam of light. However, thistechnique is subject to many limitations, including sensitivity tofluctuations in the speed of the particles and variations from therelative orientation of the particles, and only yields a measurement ofone axis of the three-dimensional particle.

[0007] Still other indirect measurements estimate nuclear volume basedon the cross-sectional areas of the nucleus. But these measurements, inturn, can be limited by variability in the staining and mountingtechniques used on the nuclei. In particular, confocal microscopes havebeen used to measure the area of stained DNA in the nucleus, summing upsuccessive cross-sections to obtain a measure of the total DNA. Byassuming that the nuclear volume is proportional to the stained DNA,this technique then yields an estimate of the nuclear volume. However,this technique fails to account for the granularity of DNA within in thenucleus, and ignores the varying contribution to nuclear volume from theother components of the nucleus: RNA, nuclear proteins, nuclear lipids,nuclear envelope and nuclear water.

[0008] Thus, previous techniques fail to meet the need for satisfactorymeasurements of nuclear volume in combination with a useful correlationwith other measurements to characterize the condition of nuclei and thecells as a whole. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF THE INVENTION

[0009] The nucleus of a cell is a highly organized structure, allowingprecursor materials to pass through pores in the nuclear envelope intothe nucleus, and nuclear products to be transported out to thecytoplasm. The complex nuclear machinery—nucleic acids, proteins, lipidsand other components—are tightly packaged within the volume of thenucleus.

[0010] As with most organized structures in nature, the nucleus assumesa shape for compact packaging of its components for optimal efficiency.When a cell becomes diseased, such as in malignant cells, the nuclearorganization breaks down. For example, the DNA loses its ability to foldefficiently around histone proteins into organized structures callednucleosomes. The protein content also changes, as well as otherbiochemical components of the nucleus. The volume of the nucleus becomesforced to increase to accommodate this disorganization. Thus, theefficiency of this packing is a characteristic of the nucleus—and auseful indication of the condition of the cell as a whole.

[0011] The present invention provides methods and devices fordetermining the nuclear packing efficiency (NPE) of a cell by measuringthe spatial displacement of the nucleus (SDN), for example by using flowcytometry to measure electronic nuclear volume (ENV). When the method isapplied to procaryotes or viruses, the SDN can be considered the volumeof the surrounding particle, which is the procaryotic cell or the virusitself. One or more biochemical components (BCs) of the nucleus are alsomeasured, such as nucleic acids, nuclear protein, nuclear lipids ornuclear water. An NPE is then determined by correlating the valuesmeasured for BC and SDN.

[0012] A variety of techniques can be used to correlate the BC and SDNto yield an NPE. Polynomial fitting can be used, from the ratio BC/SDNto more complex expressions such asNPE=k₁(BC)^(a)/(SDN)^(b)+k₂(BC)^(c)+k₃(SDN)^(d)+k₄. Graphical methodsare particularly useful for evaluating NPEs for a population of cellsand for identifying distinct subpopulations of cells. Subpopulations canthen be characterized in terms of their geometric parameters, such asdiameter, eccentricity and gradient line slope.

[0013] Once determined, NPEs are useful for identifying cells having aphenotype of interest. For example, cells can be identified by tissuesource and by the sex and species of the organism, as well as by variousstates of differentiation and stages in the cell division cycle andapoptosis. NPEs can also identify cells having various disease states asthey differ from their normal states, particularly neoplastic cellsexhibiting aneuploidy, distinguishing among benign, malignant andmetastatic cells, thus enabling the diagnosis and prognosis of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIGS. 1a to 1 c show NPE contours (electronic nuclear volume v.DNA fluorescence) for normal human cells from surface oral epithelium(FIG. 1a), intestine (FIG. 1b) and thyroid (FIG. 1c). NPE contours forother normal human cells are shown in FIGS. 2a, 3 a and 5 a.

[0015]FIGS. 2a to 2 e compare NPE contours for human lymph node andbreast cells in various states: normal human lymph node cells (FIG. 2aand perspective view FIG. 2b), cells from a benign breast tumor (FIG.2c), cells taken from a malignant primary tumor from breast tissue (FIG.2d) and cells taken from metastasizing breast cells from the tumor shownin FIG. 2d to the lymph node (FIG. 2e).

[0016]FIGS. 3a to 3 c compare NPE contours for cells from human colon invarious states: normal colon cells (FIG. 3a), cells from a primary colontumor (FIG. 3b) and metastatic cells taken from an end point of asurgical resection performed to remove the tumor shown in FIG. 3b (FIG.3c).

[0017]FIGS. 4a to 4 f show NPE contours for human cells taken from fourcancerous tissue sources: gastric (FIG. 4a and perspective view FIG.4b), prostate (FIG. 4c), ovarian (FIG. 4d and perspective view FIG. 4e)and lung (FIG. 4f).

[0018]FIGS. 5a to 5 c illustrate NPE contours for human lymphocytes invarious states: normal lymphocytes (FIG. 5a), leukemic lymphocytes (FIG.5b), and activated lymphocytes (FIG. 5c)

[0019]FIG. 6a shows an NPE contour for cells from mouse cell line P388.FIG. 6b shows NPE contours for normal and apoptotic cells from aWEHI-231 murine B lymphoma cell line (FIG. 6b and perspective view 6 c).

[0020]FIG. 7 shows a block diagram for a device for performing themethod of the invention.

[0021] In the figures, the following reference numbers are used:

[0022]1 trout red blood cell nuclei (TRBC) internal standard

[0023]2 diploid G₀/G₁ cluster

[0024]2 a Cluster of diploid G₀ cells

[0025]2 b cluster of diploid G₁ cells

[0026]2 c cluster of aneuploid G₀ cells

[0027]2 d second cluster of aneuploid G₀ cells

[0028]2 e cluster of activated lymphocytes

[0029]2 f cluster of apoptotic cells

[0030]3 diploid cells in S phase

[0031]3 c cluster of aneuploid S cells

[0032]4 diploid G₂+M cluster

[0033]4 a cluster of diploid G₂ cells

[0034]4 b cluster of diploid M cells

[0035]4 c cluster of aneuploid G₂+M cells

[0036]5 normal diploid NPE line

[0037]5 c aneuploid NPE line

[0038]5 d second aneuploid NPE line

[0039]5 f apoptotic NPE line

[0040]6 gradient line for cluster

[0041]6 e gradient line for activated lymphocytes

[0042] Not all clusters may be visible in a given contour due to theparticular threshold values selected for display and printout.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The components of the nucleus of a cell are packaged within thevolume of the nucleus. The efficiency of this packing is acharacteristic of the nucleus—and a useful indication of the conditionof the cell as a whole. Thus, the present invention provides a methodfor determining the nuclear packing efficiency (NPE) of a cell. Themethod measures the spatial displacement of the nucleus (SDN) and one ormore biochemical components (BCs) of the nucleus. The NPE is thendetermined by correlating the values of SDN and the BCs.

[0044] “Spatial displacement of a nucleus” (SDN) as used herein meansthe volume of space that is occupied by the nucleus. By occupying thatvolume, the nucleus can be said to displace any non-nuclear matter thatwould have otherwise occupied that space.

[0045] “Nucleus” as used herein generally means the organelle surroundedby the cytoplasm of the eucaryotic cell that contains the chromosomalDNA. The term encompasses the inner and outer walls of the nuclearenvelope and their associated proteins.

[0046] In addition to eucaryotic cells, the NPE can also be applied toprocaryotic cells, which lack a nucleus in the eucaryotic sense. In thiscontext the term “nucleus” is therefore used to refer to the particlesize of the entire procaryotic cell itself. Thus, references to“nuclear” volume or spatial displacement can refer to the volume orspatial displacement of the procaryotic cell. Similarly, biochemicalcomponents of the “nucleus” can also refer to cellular components ofprocaryotic cells.

[0047] Furthermore, the NPE can be applied to viruses. Because virusesare not considered cells, the term “nucleus” in this context can referto the particle size of the entire virus or of a portion such as acapsid. Thus, references to spatial displacement volume of a “nucleus”and biochemical components of the “nucleus” in the context of virusesshould be understood to refer to the volume and components of the viralparticle as a whole. Volume determination for viruses is well known inthe art (DeBlois and Wesley, J. Virol. 23:227-233 (1977).

[0048] The electronic cell volume (ECV) method is a particularly usefulmethod for measuring SDN, yielding an “electronic nuclear volume” (ENV).As used herein, the term “ENV” means the volume of the nucleus asdetermined by an ECV method. When determining ECV, particles such asnuclei are suspended in a conducting fluid, which is passed through asmall aperture. An electric field, either DC or AC, is then appliedacross the aperture, causing current to flow through the aperture. Whena particle passes through the aperture, the current is disrupted,causing a measurable pulse in the current. This pulse can be used tocount individual particles as they pass through the aperture. Thedimensions of the pulse can also be related to the size of the particle.

[0049] Refinements of the basic ECV method include shaping the inlet andoutlet volumes of the measuring aperture to reduce edge effects and toproduce a linear relationship between the measured change in current andthe volume of the particle. A particularly useful method for measuringENV is described in U.S. Pat. No. 4,818,103, to Thomas, which describesan improved flow cytometry aperture.

[0050] Compared to previous methods, such as forward angle lightscatter, the ECV method is not significantly affected by the shape ofthe particles as they are carried in the fluid. This is particularlyimportant when the particles can be nonspherical, such as biologicalparticles injected into the center of the stream. A useful method uses atime-of-flight (TOF) signal to account for such changes, as described inU.S. Pat. No. 4,298,836 to Groves. The TOF measurements can be takenfrom the optical fluorescent pulse generated by the nucleus as ittraverses an excitation light beam, or directly from the ECV pulse. Thewidth of the pulse is related to the long axis of the particle.

[0051] An SDN can be expressed in terms of volume such as cubic micronsor cubic millimeters. An SDN can also be expressed in terms of a signalgenerated from an ECV device, whether analog or digital. In the Figuresand Examples below, the ENV may be expressed as an 8-bit value,providing 256 channels of resolution. Nevertheless, the conversion fromchannels to cubic microns is provided in each figure. However expressed,the SDN is then correlated with BC measurements to determine the NPE. Itshould also be noted that the method does not require that the SDN andthe BCs be measured in any particular order.

[0052] “Biochemical component” (BC) as used herein means anyidentifiable substance in the cell that can be quantitated in physicalterms. For example, a BC can be measured in terms of amount, volume orarea, such as surface or cross-sectional area. Examples of BCs includevarious nucleic acids such as DNA and RNA, proteins, lipids and nuclearwater, as well as mixtures and subsets of individual BCs.

[0053] DNA in a nucleus can be conveniently measured by flow cytometrymethods. Flow cytometry methods are generally described in M. G.Ormerod, Flow Cytometry (BIOS Sci. Pubs., 2nd ed. 1999, and referencedcited therein). When nuclei are treated with a DNA stain, such as afluorescent stain, the quantity of DNA in the nuclei can be measured bydetecting the amount of staining or fluorescence. DNA stains includepropidium iodide, acridine orange, ethidium bromide, quinacrine,mithramycin, chromomycin A3 and 4′,6-diamidino-2-phenylindole (DAPI)(see Krishan, J. Cell Biol. 66:188-193. (1975); Darzynkiewicz et al.,Cytometry 5:355-363 (1984); Kapuscinski, Biotechnic & Histochem.70(5):220-233 (1995)) (see Example I). Automated microscopes thatmeasure absorption or fluorescence can also be used with DNA staining tomeasure the quantity of DNA in the cell nucleus (Tanke et al., J.Histochem. Cytochem. 27:84-86 (1979); Bjelkenkrantz, Histochemistry79(2):177-91 (1983)).

[0054] Indirect measurements of DNA are possible by measuring theproteins and other components associated with DNA structures in thenucleus. Chromosomal DNA is coiled around octomers of histone proteins(two each of H2A, H2B, H3 and H4) to form a complex termed a nucleosome.Together with nonhistone chromosomal proteins, DNA-histone structuresform fibers collectively termed chromatin, which is itself divided intometabolically active euchromatin and transcriptionally inertheterochromatin. Measurement of any of these DNA-associated proteins cantherefore be a useful measure of DNA content. For example, labeledantibodies that specifically bind to any of these proteins areparticularly useful for such indirect measurements of nuclear DNA. Inparticular, histones be measured by flow cytometry and automatedmicroscopes using antibodies against specific histones (Miller et al.,Hybridoma 12(6):689-698 (1993)). Histones can also be quantitated byelectrophoretic examination using a silver stain (Tsutsui et al., Anal.Biochem. 146(1):111-117 (1985)).

[0055] RNA has similarly been measured with flow cytometry usingacridine orange staining (Piwnicka et al., Cytometry 3(4):269-75(1983)). RNA can also be measured by cytophotometric image analysisusing methyl green-pyronin Y stain (Schulte et al., Histomchem. J.24(6):305-310 (1992)). As with DNA, RNA-related proteins, such asnuclear proteins related to RNA transcription, can be used to measureRNA indirectly.

[0056] The nucleolus is another nucleic-acid-related BC, a largestructure where large numbers of rRNA copies are transcribed andimmediately packaged with ribosomal proteins to form ribosomes. Thus,antibodies against any of the nucleolus machinery can provide a usefulBC for measurement.

[0057] Lipids that are BCs include any measurable lipids in the nucleussuch as nuclear envelope lipid, whether in the inside or outside walls.These lipids can be measured directly or indirectly by measuringnuclear-envelope-associated proteins. Outer wall nuclear membraneproteins include ribosomes. Proteins associated with the inner wall ofthe nuclear membrane include nuclear pore proteins, nuclear laminaproteins and lamina-associated polypeptides. Fluorescent lipophilic dyes(Collas et al., Dev. Biol. 169(1):123-35 (1995)) and monoclonalantibodies that recognize the nuclear pore antigens (Matsouoka et al.,Biochem. Biophys. Res. Commun. 254(2):417-423 (1999)) can be used tomeasure various components of the nuclear envelope and to provide ameasure of lipid content, as well.

[0058] Although proteins have been discussed as a means to indirectlymeasure nucleic acids and lipids, nuclear proteins can be BCs in theirown right. Nuclear proteins can be detected by nonspecific stainingusing FITC fluorescence in combination with flow cytometry (Roti et al.,Cytometry 3(2):91-96 (1982)). Another method is to measuredinitrofluorobenzene (DNF) absorbance using cytophotometric imageanalysis (Cohn et al., Histochemistry 79(3):353-364 (1983). Yet anothermethod is to measure protein-bound sulfhydryl groups with thefluorescence of AEDANS (Schabronath et al., Cytometry 11(3):333-340(1990)).

[0059] Nuclear Matrix Proteins (NMPs) and other specific nuclearproteins can also be measured as BCs. NMPs can be measured usingantibodies prepared against purified protein molecules located in thenuclear matrix. As with the antibodies discussed above, they can then beconjugated with fluorescent stains and used with flow cytometry orimmuno-histochemical staining to detect specific NMPs (Hughes et al.,Am. J. Clin. Path. 111(2):267-274 (1999).

[0060] Nuclear water and associated non-organic salts make up theremainder of the volume in the nucleus. Nuclear water can be measured byENV when performed at isotonic and iso-osmotic conditions.

[0061] As disclosed above, an NPE is determined by correlating the SDNand BC values. This correlation may be achieved by a variety ofmathematical functions and operations. For example, the NPE can bedetermined by using a general polynomial function such as

NPE=k ₁(BC)^(a)/(SDN)^(b) +k ₂(BC)^(c) +k ₃(SDN)^(d) +k ₄.

[0062] In this formula, k₁, k₂, k₃, k₄, a, b, c and d are individuallypreselected constants and k₁ is not zero. Particularly useful values fork₁, k₂, k₃, k₄, a, b, c and d are 2, 1, ½, 0, −½, −1, and −2independently, with the proviso that k₁ is not zero. A specificapplication of the general formula is where each of k₁, a and b are 1and each of k₂, k₃ and k₄ are zero, resulting in the ratio

NPE=BC/SDN.

[0063] Determination of an NPE is not limited to measuring andcorrelating a single BC, however.

[0064] A second biochemical component (BC₂) can be measured as a furtherstep in the method. The BC₂ can be any of the BCs disclosed above, suchas nucleic acids, lipids, proteins and nuclear water, so long as it isdifferent from the first BC. BC₂ can then be incorporated into thedetermination of NPE by a variety of formulas using the expressionk₅(BC₂)^(e), where k₅ and e are each preselected constants such as 2, 1,½, 0, −½, −1 and −2. For example, k₅(BC₂)^(e) can be added to the valueof BC in the general formula, so that the general formula becomes

NPE=k ₁(BC+k ₅(BC ₂)^(e))^(a)/(SDN)^(b) +k ₂(BC+k ₅(BC₂)^(e))^(c) +k₃(SDN)^(d) +k ₄.

[0065] Similarly, k₅(BC₂)^(e) can be added to the value of SDN in thegeneral formula to obtain

NPE=k ₁(BC)^(a)/(SDN+k ₅(BC ₂)^(e))^(b) +k ₂(BC)^(c) +k ₃(SDN+k ₅(BC₂)^(e))^(d) +k ₄.

[0066] Furthermore, the NPE obtained by the general polynomial formulacan be multiplied by k₅(BC₂)^(e):

NPE=k ₅(BC ₂)^(e)×(k ₁(BC)^(a)/(SDN)^(b) +k ₂(BC)^(c) +k ₃(SDN)^(d) +k₄).

[0067] The polynomial expressions above are merely intended toillustrate the range of useful correlations possible between BCs andSDN. Specific examples of NPE correlations include the following:

NPE=DNA/ENV

NPE=(fluorescence of DNA)/ENV

NPE=DNA/ENV ^(1/2)

NPE=DNA/(procaryotic cell volume)

NPE=RNA/(volume of viral particle)

NPE=NMP/ENV

NPE=(mass of nucleosome)/ENV

NPE=(volume of nuclear envelope)/ENV

NPE=(DNA−RNA)/ENV

NPE=(DNA+RNA)/ENV ²

NPE=(DNA×RNA)/ENV ²

NPE=DNA/(ENV−(nuclear water))

NPE=(DNA+nuclear matrix protein)/ENV

NPE=DNA ⁻¹/(ENV+RNA)⁻¹

NPE=(DNA+RNA)/ENV ²

NPE=2(DNA)² /ENV+0.5(DNA)−ENV ²+5

NPE=(DNA+RNA)² /ENV−(DNA+RNA)^(−1/2) +ENV

[0068] Mathematical variations of these correlations between BC and SDNcan include trigonometric, logarithmic and exponential and othertranscendental functions while not falling within the strict formula ofthe polynomial itself. These variations should be considered equivalentso long as they mathematically relate BC and SDN in a substantiallysimilar way to achieve a substantially similar correlation that is auseful measure of NPE. Thus, determining NPE is not limited tofunctions, however, but can be determined using a number of moresophisticated methods.

[0069] Graphical methods provide a powerful tool for determining andvisualizing NPEs (see Examples II.D and III). As an example, a BC andSDN can be measured for a cell and plotted on separate axes for BC andSDN, resulting in a datapoint for the cell. The term “datapoint” meansherein any mathematical correlation between two or more values. Thus, adatapoint for a cell can also correlate DNA, RNA and SDN. The term canrefer to a graphed or plotted point, but can also refer to anyrepresentation of the values of BC and SDN, whether displayed visually,represented numerically or stored in a computer's memory. However, eachdatapoint's values should represent a single cell or small group ofcells.

[0070] Datapoints for multiple cells can also be plotted, eitherseparately or on the same plot. If on the same plot, the datapoints canbe represented by a variety of graphical methods. Scattergrams simplyshow each datapoint on the same graph, where the density of datapointsreflects the number of cells having BCs and SDNs of a certain value orrange. Contour plots can be used illustrate the frequency of datapointsin a certain range by plotting a contour or surface, where a separateaxis is used to indicate frequency.

[0071] An example of a graphical representation of datapoints is FIG.2b, which shows data from normal human lymph node. The horizontal axisis DNA fluorescence, reflecting the amount of DNA in each nucleus, andthe depth axis is ENV, reflecting nuclear volume. The vertical axis isthe number of cells having a datapoint at each given value on thehorizontal and depth axes. Thus, a frequency contour is provided for ENVv. DNA.

[0072] As shown in this perspective view, the majority of the lymph nodecells are in a single peak representing a cluster of cells 2 that arediploid G₀/G₁ cells. The second highest peak represents a cluster oftrout red blood cell nuclei (TRBC) 1 added to the sample as an internalcontrol (see Example I.C.). A much smaller peak is also visiblerepresenting a cluster of diploid G₂+M cells 4 in the sample of lymphnode cells.

[0073] The same data can be represented in a contour graph. In FIG. 2a,the horizontal axis is DNA fluorescence and the vertical axis is ENV.The frequency of cells having each ENV v. DNA data point is shown bycontour lines, representing lines of equal frequency, much as atopographic map uses lines of equal elevation. As before, the cluster ofTRBC internal standard 1 is seen, as well as clusters representing thetwo subpopulations of lymph node cells: diploid G₀/G₁ cells 2 anddiploid G₂+M cells 4.

[0074] Surprisingly, a line 5 can be drawn from the origin through thecenters of the clusters 2 and 4. This is because the two clusters sharea common correlation between ENV and DNA, even though the individualvalues for ENV and DNA differ by about two-fold. In short, the twoclusters of lymph node cells share a common NPE that is characteristicfor lymph node cells. In graphical terms, where a line has the formulaBC=NPE(SDN), the slope of the line (NPE) is BC/SDN. Alternatively, theline can have the formula SDN=NPE(BC), where the slope (NPE) is SDN/BC.A line or substantially linear curve representing NPE can be determinedgraphically by hand, by calculator or determined by a variety ofmathematical and statistical software packages. Thus, an NPE can bedetermined graphically for datapoints representing multiple cells.

[0075] An added advantage to representing the datapoints graphically isthat distinct clusters of datapoints can be identified, eachrepresenting subpopulations of cells in the original sample. For examplein FIG. 2a, clusters 2 and 4 are readily identified with separatesubpopulations of cells. The term “cluster” means herein any subset ofthe entire set of datapoints plotted for a population of cells or for arepresentative number of cells in a preselected population. Similarly,the term “subpopulation” means herein the cells represented by thedatapoints in a cluster. Thus, in FIG. 6a, separate subpopulations ofcells can be discerned by clusters 2 a, 2 b, 3, 4 a and 4 b. It shouldbe noted, however, that when virtually all the datapoints form awell-defined group, cluster 2 in FIG. 1a for example, the terms clusterand subpopulation may still apply.

[0076] More formally, a cluster can be defined as a neighborhood ofdatapoints that are adjacent to a local maximum of datapoints andcharacterized by decreasing frequency as the distance from the localmaximum increases. If desired, a frequency threshold or cut-off can beused to further resolve the separation between clusters, and thesubpopulations represented by the clusters.

[0077] Geometric parameters can then used to describe thecharacteristics of each cluster. The term “geometric parameter” meansherein any geometric or mathematical property of a cluster. The centerof a roughly circular cluster is a parameter that can be defined as theaverage center point, the centroid or the local maximum. The slope of aline passing through the origin and the center of a cluster is aparticularly useful parameter. Where the shape of the subpopulationcontour is ellipsoid, other useful geometric parameters include theeccentricity, the maximum range of the major axis, the maximum range ofthe minor axis and the standard deviations of the major and minor axes.Another useful geometric parameter is the perimeter of the cluster whenrepresented graphically at a predetermined threshold value.

[0078] Other mathematical variations of these geometrical parameters forclusters can include a variety of polynomial, trigonometric,logarithmic, exponential and other transcendental functions, while notfalling within the list of parameters described above. These variationsshould be considered equivalent so long as they mathematically describea geometrical feature of the cluster in a substantially similar way toachieve a substantially similar description that is a useful descriptionof the cluster.

[0079] A gradient line is another particularly useful geometricparameter, indicating the general tilt of an ellipsoid or elongatedcluster. The term “gradient line” means the line passing orthogonallythrough the direction of highest slope in a cluster. Examples ofgradient lines are the lines marked 6 in FIG. 2d. The line orthogonal tothe gradient line can also be a useful geometric parameter of a cluster.Although a gradient line can often be hand-drawn by inspection, the linecan be determined more precisely by performing a linear regression ofthe datapoints in the subpopulation.

[0080] Having generated a contour of datapoints and identified clustersrepresenting subpopulations of cells in the sample, the geometricparameters identified can then be used to identify different cellswithin a population of cells by identifying the cell if the cell's NPEis within at least one predetermined NPE range. Separately orconcurrently, the method can further involve segregating the identifiedcell from non-identified cells. The term “segregating” herein means toseparate the cells into distinctly separate areas or containers and arenot in their original state or in a uniform mixture.

[0081] It should be emphasized that the methods for determining an NPEfor a population of cells are not limited to graphical, but can beperformed equally well numerically. Thus, each of the steps of thedisclosed method for determining an NPE for a population ofcells—including determining a datapoint for BC and SDN, identifying acluster of datapoints and determining an NPE according to a preselectedgeometric parameter—can be performed numerically. For example, thesesteps can be performed by a computer without necessarily representingthe data in graphical form. Thus, disclosed applications for NPEs andNPE contours should be understood to apply equally to both.

[0082] NPEs and NPE contours can be used to identify cells having aphenotype of interest. Cells can be identified by comparing their NPEswith other NPEs. For example, an NPE of a cell of interest can becompared with a predefined range of NPEs for a reference population ofcells, or against other cells in the sample. As a result, NPEs and NPEcontours are a useful characteristic to identify the phenotype of cellsin a sample.

[0083] A particular use for NPEs is to determine the sex of an organismby its cells. In the somatic cells of many species, cells from femaleanimals have two X chromosomes, while cells from male animals have one Xand a smaller Y chromosome. As a result, male cells have less DNAcontent than female cells, but with comparable nuclear volumes. Thisdifference is reflected by a lower packing efficiency. In FIG. 2a,cluster 2 tends to have a lower DNA content (DNA fluorescence channelcloser to 76) when cells are from males and a higher DNA content (DNAfluorescence channel closer to 80.5) when the cells are from females.These changes are reflected in a higher slope (decreased NPE) for malesand lower slope (increased NPE) for females. Thus, cells from animals ofdifferent sexes can be distinguished. The method is equally applicablein other animals, such as certain waterfowl, where the sex chromosomesare reversed.

[0084] NPEs can also be used to determine whether cells are fromdifferent tissues. For example, FIGS. 1a, 1 b, 1 c, 2 a, 3 a and 5 ashow NPE contours for cells from different tissues, each havingcharacteristic NPEs. Thus, cells of unknown origin can also beidentified by comparing their NPEs with NPEs of known tissues.Similarly, cells in different stages of differentiation can beidentified by their NPEs (see Example III.C.).

[0085] A further use for NPEs is to determine whether cells are fromdifferent species. In FIG. 1a, for example, the NPE for nuclei fromtrout are distinguished from an NPE for human lymph cells. Similarly,the NPEs of the clusters in FIG. 6, representing cells from a mouse cellline, are distinguishable from the NPE in FIG. 1a. Thus, cells fromdifferent species can be distinguished by their NPEs.

[0086] Cells in different stages of the cell division cycle can also beidentified using NPE methods. Normally dividing cells undergo awell-defined series of stages to coordinate cell division into twodaughter cells and the corresponding replication of DNA necessary tomaintain a complete set of chromosomes for the daughter cells. During S(“synthesis”) phase, a cell replicates its nuclear DNA, doubling the DNAcontent of the nucleus. After resting during G₂ phase, the nucleusdivides during M (“mitosis”) phase, evenly separating the replicatedDNA, followed by cytokinesis, where the cell itself divides intoseparate daughter cells. Cell division is then followed by the G₀ and G₁phases before initiating S phase again. As shown in Example III.D, NPEscan be used to identify cells in different stages of the cell cycle.

[0087] Because the NPE of a cell is maintained during the cell cycle sorigorously, it follows that disruptions of the condition and normalgrowth of a cell will be reflected in its NPE. Thus, the NPE can be usedto identify cells in an apoptotic state, when the cells undergoapoptosis, programmed cell death (see Example III.E).

[0088] Furthermore, NPEs can be used to identify an abnormal conditionssuch as a pathology or disease state. Disease states include geneticdiseases such as sickle cell anemia. For example, the extra chromosomespresent from Down's syndrome and Klinefelter's syndrome can be detectedusing NPEs. Similarly, genetic anomalies associated with autoimmunediseases can also be detected.

[0089] NPEs are particularly useful for identifying a neoplastic state.As used herein, the term “neoplastic” means characterized by formationand growth of abnormal tissue that grows more rapidly than normal.Neoplastic tissue can show partial or complete lack of structuralorganization and functional coordination with normal tissue. Neoplasticcells are often characterized by aneuploidy. The term “aneuploidy”herein means having an abnormal number of chromosomes. Aneuploid cellsare contrasted with cells having a normal number of chromosomes,although the precise number can vary depending on the stage of divisionin the cell division cycle. Thus, the method can be useful fordistinguishing neoplastic cells from normal tissue.

[0090] As discussed in Example III, normal cells (see FIG. 2a) can becharacterized by relatively circular clusters, while neoplastic cellscan be characterized by relatively elongated clusters (see FIGS. 2c, 2d, and 2 e). The clusters can become elongated due to the presence ofaneuploid cells, labeled 2 c, 2 d and 4 c. In turn, these clusters ofaneuploid cells can give rise to one or more aneuploid NPE lines 5 c, 5d (see FIGS. 2d, 2 e).

[0091] Among neoplastic cells, benign tumors can have clusters withrelatively vertical gradient lines 6 (see FIG. 2c), while cells frommalignant primary tumors (see FIGS. 2d and 3 b) can have clusters withtilted gradient lines 6. In addition, if metastasis has occurred, theprimary tumor clone can be identified by its tilted gradient line andits NPE in the metastatic site (see FIGS. 2d, 2 e, 3 b, 3 c).

[0092] Furthermore, neoplastic cells can be characterized by havingabnormal ratios of cells in the G₀/G₁ cluster 2 and G₂+M cluster 4. Forexample, G₀/G₁ cells 2 clearly predominate over the G₂+M cells 4 in thenormal human lymph node cells shown in FIG. 2b. In the ovarian cancercells in FIG. 4e, however, there are nearly equal numbers of G₀/G₁diploid cells 2 and G₂+M diploid cells 4, indicating that the ratio ofcells in the G₂ and M stage of the cell division cycle has becomeabnormal. Thus NPEs can be useful for identifying a neoplastic state ina perspective view.

[0093] These methods are applicable to any type of tissue subject tocancer, including lymph node (FIG. 2e), breast (FIGS. 2c, 2 d), colon(FIGS. 3b and 3 c), gastric (FIG. 4a), prostate (FIG. 4c), ovarian (FIG.4d), lung (FIG. 4f), leukemia (FIG. 5b), as well as cervical andtesticular tissue, pancreas, liver, brain and small intestine. Othertissues subject to these methods include brain, ovary, testes, bone andexfoliated circulatory tissue.

[0094] Devices for performing the methods disclosed above are alsoprovided by the present invention. A device for determining the NPE of acell can have means for measuring one or more BCs and measuring SDN, andmeans for determining the NPE. A block diagram of such an embodiment ofthe device is shown in FIG. 7. Specific measuring means are discussed indetail above, in the examples below, and in the publications and patentscited herein.

[0095] Each of the publications and patents cited herein are herebyincorporated by reference. The term “comprising” as used herein,including its use in the body of the claims, is intended to beopen-ended, thereby encompassing the recited elements or steps, as wellas encompassing embodiments having additional elements or steps. Thefollowing examples are intended to illustrate but not limit theinvention.

EXAMPLE I Preparation of Stained Nuclei

[0096] The following examples illustrate the preparation of isolatednuclei from tissue samples.

[0097] A. From Solid Tissue

[0098] Isolated nuclei from solid tissue specimens were prepared asfollows. Approximately 1 to 2 cubic millimeters of tissue was placed ina petri dish with 2 ml of the following nuclear isolation solution toisolate and stain the nuclei: 10 mg/ml 4′,6-diamidino-2-phenylindole(DAPI) DNA stain (Sigma Chemical Co.; St. Louis, Mo.); 0.6% NP-40 (v/v)(Accurate Scientific & Chemical Co.; Hicksville N.Y.); in isotonicphosphate buffered saline solution at pH 7.2. The isolated stainednuclei were filtered through a 35 micron polypropylene filter (RATCOM,Inc.; Miami Fla.) and placed on ice. Using this method, samples ofisolated, stained nuclei were prepared from a variety of solid tissuesources, including lymph node, intestine, breast, colon, thyroid, ovary,prostate, stomach and surface epithelial cells from mouth.

[0099] B. Human Lymphocytes

[0100] Human lymphocytes were isolated from venous whole blood usingficoll hypate, washed with isotonic saline, and adjusted to aconcentration of about 1×10⁷ lymphocytes/ml. Then, 0.1 ml of thislymphocyte solution was added to 1 ml of the nuclear isolation solutiondescribed above, resulting in a solution of lymphocyte nuclei at aconcentration of approximately 1×10⁶ nuclei/ml. The stained nuclei werefiltered through a 35 micron polypropylene filter and placed on ice.

[0101] C. Trout Red Blood Cell Nuclei (TRBC)

[0102] Nuclei from trout red blood cells were used as an internalstandard. Trout (Salmo gairdnerii irideus) red blood cells (U.S. FishHatchery; Erwin Tenn.) were prepared into stock solutions as follows:150 μl of a commercially available TRBC solution. (RATCOM Inc.; MiamiFla.) was added to 2 ml nuclear isolation solution described above,resulting in a stock solution of TRBC nuclei at a concentration of about2×10⁶ nuclei/ml. The stained TRBC nuclei were then filtered through a 35micron polypropylene filter and placed on ice.

EXAMPLE II Determination of NPEs

[0103] A. DNAnalyzer Flow Cytometer

[0104] The DNAnalyzer™ flow cytometer (RATCOM Inc.; Miami Fla.)simultaneously analyzed the DNA content by fluorescence and the volumeof each particle (ENV) as it passed through the measuring aperture ofthe instrument. The volume was determined by the Coulter Electronic CellVolume principle described in U.S. Pat. No. 2,656,508 to Coulter, andaccording to the teaching in U.S. Pat. No. 4,818,103 to Thomas andEggleston.

[0105] The DNAnalyzer used a unique equilateral triangle flow cell witha cross-section of 70 microns per side and 70 microns in length. Theinlet and outlet were also equilateral triangles in cross-section, withdimensions starting at 1 cm per side, and decreasing in size over 1 cmto 70 microns per side. The instrument employed epi-illumination andcollection optics (i.e. collection optics from the same side of thesample as excitation optics) with a 410 nm dichroic mirror to excite thesample and collect the fluorescence emission.

[0106] The excitation source was a 100-watt stabilized mercury arc lampwith a 30-micron spot size in the most uniform region of the arc. A UG1filter selected the optimal excitation wavelength for the DAPI of 365 nmfrom the emission of the mercury arc lamp. The micro-objective had a1.25 N.A. corresponding to a collection angle of 120 degrees forcollection of the fluorescence emission. The DNAnalyzer then usedDigital Pulse Processing (DPP) to determine the peak value of thefluorescence emission and nuclear volume from each nucleus.

[0107] B. Sample Analysis

[0108] Samples of stained nuclei prepared as described in Example I wereanalyzed as follows. Approximately 50 μl of the TRBC stock suspensionwas added to each sample as an internal DNA standard. The sample wasthen analyzed on the DNAnalyzer at a flow rate of 60 to 120nuclei/second and at least 15,000 events were collected. The data werecollected at 8 bits of resolution for both ENV and DNA fluorescence, andpresented in an FCS 2.0 standard file format (The Institute ofElectrical and Electronics Engineers, Inc; New York N.Y.).

[0109] The data were analyzed using Modfit version 5.11 (Verity SoftwareHouse Inc.; Topsham Me.). The two-parameter graphs were prepared usingWinMDI version 2.7 (freeware available at http://facs.scripps.edu). Thecontour and perspective plots were analyzed in log mode with theinterval set at 85%, smooth at 6, and threshold at 0.5. The displayresolution was 256×265 for the contour and perspective displays.

[0110] Calibration was performed for volume using 4 micron calibrationspheres and the known value for trout RBC nuclei of 5 pg of DNA per TRBCnucleus.

[0111] C. Determination of NPEs

[0112] Normal cells were prepared as described in Example I and measuredfor nuclear volume (cubic microns) and DNA content (picograms DNA). NPEswere determined in terms of DNA/ENV: tissue type pg DNA cu. micron NPElymphocyte 7.75 21.3 0.364 lymph node 7.60 21.8 0.348 breast 8.14 23.60.345 colon 7.90 22.2 0.356 intestine 7.81 21.8 0.358 thyroid 8.29 24.30.341

[0113] Alternative formulas for NPEs were also determined: tissue typeENV/DNA DNA²/ENV (DNA + ENV)/ENV lymphocyte 2.75 2.82 1.364 lymph node2.87 2.65 1.348 breast 2.90 2.81 1.345 colon 2.81 2.81 1.356 intestine2.79 2.80 1.358 thyroid 2.93 2.83 1.341

[0114] D. Graphical Methods for Determining NPEs

[0115] Examples of NPE contours for normal human tissue are presented inthe figures for surface epithelial tissue from mouth (FIG. 1a),intestine (FIG. 1b), thyroid (FIG. 1c), lymph node (FIG. 2a), colon(FIG. 3a) and lymphocytes (FIG. 5a). A perspective view of FIG. 2a isprovided as FIG. 2b to emphasize the relative heights of the peaks.

[0116] In each of the figures, internal standard TRBC appears as cluster1. The values for cubic microns and picograms of DNA per channelaccompany each of the axes. A line 5 passes through the origin and theG₀/G₁ peak 2 and any diploid G₂+M peak 4 visible. An NPE (ENV/DNA) canthen determined to be the slope of the line 5. Alternatively, theinverse slope (DNA/ENV) can be used as the NPE.

EXAMPLE III Using NPEs to Characterize Cells

[0117] This example illustrates the use of NPEs to characterize cells,for example by distinguishing among normal and neoplastic cells, andcells in different stages of differentiation and apoptosis.

[0118] A. Human Lymph Node Cells

[0119] For reference, FIG. 2a shows an NPE contour for cells from normalhuman lymph node. The G₀/G₁ cluster 2 and diploid G₂+M cluster 4 arerelatively circular and their centers are aligned on NPE line 5.

[0120] In contrast to FIG. 2a, FIG. 2c shows an NPE contour for cellsfrom benign breast tumor. The normal cells 2, 4 have an NPE of about0.145 (pg DNA/cu. microns), shown as normal NPE line 5. As shown, theshape of the G₀/G₁ cluster 2 has stretched vertically by the presence ofaneuploid cells 2 c. At the upper edge of the 2 c cluster, the NPE canbe as low as 0.023 (pg DNA/cu. microns). This change in the shape of thecluster is indicative of a neoplastic state. It should be noted that thegradient line 6 is essentially vertical in this figure, expressible as anearly vertical slope of 1041 (change in volume/change in DNA). Also,the TRBC internal standard 1 is also stretched somewhat as an artifact,due to adhesion of foreign particles to the TRBCs.

[0121]FIG. 2d shows an NPE contour for cells from a malignant primarybreast tumor. As shown, the G₀/G₁ cluster 2 and diploid G₂+M cluster 4are both elongated by aneuploid cells 2 c and 2 d. Significantly, thegradient lines 6 are tilted clockwise from the vertical, expressible asa relatively less vertical slope of 9.4. Thus, a change in the slope ofthe gradient line of a cluster can be indicative of an abnormalcondition such as a malignant neoplastic state.

[0122]FIG. 2e shows an NPE contour for metastatic cells from the primarybreast tumor in FIG. 2d. The two aneuploid clusters 2 c and 2 d fromFIG. 2d are clearly recognizable by the slope of the clusters, gradientslope and NPE slopes in the metastatic aneuploid populations 2 c and 2 din FIG. 2e. Thus, a change in the width, breadth or shape of the peakcan be a significant indication of an abnormal cell condition such asmetastasis. Moreover, FIG. 2e shows that aneuploid population 2 d can beclearly discerned from the diploid G₂+M population 4.

[0123] Furthermore, in FIG. 2e, the second aneuploid population 2 daccounts for 3.4% of the cells, compared to 0.9% of the cells in thediploid G₂+M population 4, showing a high ratio of second aneuploidcells compared to G₂+M cells. Previously, tetraploidy could only beconfirmed when the G₂+M population was greater than 15%. Here, thisratio can be observed with G₂+M levels lower than previously known inthe field and could previously be observed from a profile of DNAfluorescence alone (see Hankey et al., Cytometry 14:472-477 (1993)).

[0124] B. Human Colon Cells

[0125]FIG. 3a shows an NPE contour for cells from normal human colon.

[0126]FIG. 3b shows an NPE contour for cells from a primary tumor.Significantly, the G₀/G₁ cluster 2 has become elongated by aneuploidcells 2 c, and has a tilted gradient line 6. An aneuploid NPE line 5 cis shown passing through the cluster of aneuploid cells 2 c

[0127]FIG. 3c shows an NPE contour for cells taken from at the end pointof resection when surgically removing a metastasizing tumor shown inFIG. 3b. As shown, the aneuploid cells 2 c are still present and have atilted gradient line 6, indicating the resection sample containsmetastatic cells.

[0128]FIGS. 4a to 4 f show NPE contours for other cancerous tissuesources: gastric (FIG. 4a and perspective view FIG. 4b), prostate (FIG.4c), ovarian (FIG. 4d and perspective view FIG. 4e) and lung (FIG. 4f).Notably, FIG. 4f of lung cancer cells shows a cluster of aneuploid Scells 3 c and a cluster of aneuploid G₂+M cells 4 c (TRBC standardomitted for scaling reasons), as well as a distinct aneuploid NPE line 5c passing through 2 c, 3 c and 4 c.

[0129] C. Human Lymphocytes

[0130]FIG. 5a shows an NPE contour for normal blood lymphocytes. TheG₀/G₁ cluster 2 is very slightly elongated vertically.

[0131]FIG. 5b shows an NPE contour for leukemic lymphocytes. As shown,the presence of aneuploid cells 2 c has altered the shape of the G₀/G₁cluster 2, resulting in a tilted gradient line 6 and an NPE 5 c for theaneuploid population clearly discernable from the diploid NPE line 5.The NPE contour provides a clear indication that aneuploid cells 2 c arepresent in the sample.

[0132]FIG. 5c shows an NPE contour for a cell sample of normal bloodcontaining activated lymphocytes. In addition to the G₀/G₁ cluster 2, anadditional cluster of activated lymphocytes 2 e is discernable, with adistinct vertical gradient line 6 e. Thus an NPE contour is useful fordetecting cells having a different state of differentiation.

[0133] D. Cell Cycle

[0134] The progress of the cell cycle can be traced in FIG. 6a, whichshows a contour of datapoints from a sample of a mouse cell linecontaining cells at various stages of the cell cycle. Cells at G₀ phaseare shown in cluster 2 a. A slight increase in nuclear volume isreflected by the cells in G₁ phase in cluster 2 b. Once the cells beginreplicating their DNA during S phase 3, the values for DNA fluorescencebegin to increase until they are double the values for cells in G₀phase, where they remain in G₂ phase 4 a. Corresponding aneuploid cellsin S phase 3 c and G₂+M phase 4 c are shown in FIG. 4f.

[0135] Upon initiating nuclear division, the cells in M phase 4 b show aslight increase in nuclear volume. Once nuclear division is complete,the amount of DNA and the nuclear volume are both halved, as shown bythe return to G₀ phase 2 a. Thus, the progress of cells through eachstage of the cell cycle is reflected in the clusters identifiable inFIG. 6a. The cell cycle can also be seen in FIGS. 2a and 2 b.Remarkably, the clusters remain aligned on the NPE line 5 throughout,demonstrating that the characteristic NPE for the cell line ismaintained, even while the nuclei and cells are dividing.

[0136] E. Apoptotic Cells

[0137] WEHI cells from a WEHI-231 murine B lymphoma cell line (LombardiCancer Institute; Wash. D.C.) were obtained untreated or treated with anapoptotic agent. As shown in FIG. 6b, the NPE contour for nonapoptoticcells contains cells in various stages of the cell cycle, as discussedabove (clusters 2 a, 2 b, 3, 4 a, 4 b). The apoptotic cells are in adistinct cluster 2 f, with a different NPE line 5 f. The separationbetween the nonapoptotic and apoptotic cells is highlighted inperspective view FIG. 6b (the origin is in the distant upper rightcorner). It can be observed that the volume of the nucleus remainsrelatively constant whether apoptotic or not, but the measured DNAdecreases during apoptosis, whether due to decrease in DNA content or inDNA staining. This suggests that the volume of the nucleus duringapoptosis is not maintained by the DNA content, but by other components,such as the nuclear matrix.

[0138] Although the invention has been illustrated by the examplesabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

We claim:
 1. A method for determining the nuclear packing efficiency(NPE) of a cell, comprising the steps of (a) measuring a biochemicalcomponent (BC) of the nucleus of a cell; (b) measuring a spatialdisplacement of the nucleus (SDN) of the cell; and (c) determining anuclear packing efficiency (NPE) by correlating the values of BC andSDN.
 2. The method of claim 1, wherein the SDN is the volume of aparticle selected from the group consisting of a procaryotic cell and avirus.
 3. The method of claim 1, wherein the SDN is the volume of aeucaryotic nucleus.
 4. The method of claim 3, wherein the SDN ismeasured using electronic cell volume (ECV) to yield an electronicnuclear volume (ENV).
 5. The method of claim 4, wherein the ENV isadjusted using flow cytometry time-of-flight (TOF).
 6. The method ofclaim 1, wherein the BC includes nucleic acid.
 7. The method of claim 6,wherein the nucleic acid is DNA.
 8. The method of claim 7, wherein theDNA is measured by fluorescence.
 9. The method of claim 6, wherein thenucleic acid is RNA.
 10. The method of claim 1, wherein the BC includesnuclear envelope lipid.
 11. The method of claim 1, wherein the BCincludes nuclear protein.
 12. The method of claim 11, wherein thenuclear protein is nuclear matrix proteins (NMP).
 13. The method ofclaim 11, wherein the nuclear protein is histones.
 14. The method ofclaim 11, wherein the nuclear protein is nuclear-envelope-associatedproteins.
 15. The method of claim 14, wherein thenuclear-envelope-associated protein is a nuclear pore protein.
 16. Themethod of claim 1, wherein the BC includes nuclear water.
 17. The methodof claim 1, wherein step (c) is performed according to the formula NPE=k₁(BC)^(a)/(SDN)^(b) +k ₂(BC)^(c) +k ₃(SDN)^(d) +k ₄; wherein k₁, k₂, k₃,k₄, a, b, c and d are preselected constants and k₁ is not zero.
 18. Themethod of claim 17, wherein k₁ is positive.
 19. The method of claim 17,wherein k₂ is zero.
 20. The method of claim 17, wherein k₄ is zero. 21.The method of claim 17, wherein k₁=1, a=1 and b=1, whereby NPE=BC/SDN.22. The method of claim 17, further comprising the step of measuring asecond biochemical component (BC₂).
 23. The method of claim 22, whereinBC₂ is selected from the group consisting of total nucleic acid, DNA,RNA, nuclear protein, nuclear matrix protein, histones, nuclear envelopelipid and nuclear-envelope-associated proteins.
 24. The method of claim22, wherein the amount of k₅(BC₂)^(e) is added to the value of BCmeasured in step (a), wherein k₅ and e are preselected constants. 25.The method of claim 24, wherein BC₂ is DNA, k₅=1 and e=1.
 26. The methodof claim 22, wherein the amount of k₅(BC₂)^(e) is added to the value ofSDN measured in step (b), wherein k₅ and e are preselected constants.27. The method of claim 26, wherein BC₂ is RNA, k₅=1 and e=1.
 28. Themethod of claim 22, wherein the NPE in step (c) is multiplied byk₅(BC₂)^(e), wherein k₅ and e are preselected constants.
 29. The methodof claim 28, wherein BC₂ is nuclear protein, k₅=1 and e=1.
 30. Themethod of claim 1, wherein step (c) is performed by performing the stepsof (c1) determining a datapoint for BC and SDN on separate axes for BCand SDN; and (c2) determining NPE as the slope of a line passing throughthe datapoint and the origin of the axes.
 31. A method for determiningan NPE for a population of cells, comprising the steps of (a) for arepresentative number of cells in the population: (1) measuring abiochemical component (BC) of the nucleus of a cell; (2) measuring aspatial displacement of the nucleus (SDN) of the cell; and (3)determining a datapoint for BC and SDN on separate axes for BC and SDN;(b) identifying at least one cluster of the datapoints; and (c)determining an NPE according to a preselected geometric parameter of thecluster of datapoints.
 32. The method of claim 31, wherein the geometricparameter is the slope of a substantially linear curve passing throughthe local maxima of at least one cluster and through the origin of theBC and SDN axes.
 33. The method of claim 31, wherein the geometricparameter is the slope of the gradient line of the cluster ofdatapoints.
 34. The method of claim 31, wherein the geometric parameteris selected from the group consisting of eccentricity, maximum range ofthe major axis, maximum range of the minor axis, standard deviation ofthe major axis, standard deviation of the minor axis, slope of a lineorthogonal to the gradient line, and perimeter.
 35. A method foridentifying different cells within a population of cells, comprising thesteps of (a) performing the method of claim 1 on at least one cell inthe population; and (b) identifying the cell if the cell's NPE is withinat least one predetermined NPE range.
 36. The method of claim 35,further comprising the step of (c) segregating the identified cell fromnon-identified cells.
 37. A method for identifying a cell, having aphenotype of interest, that is present in a population of cells,comprising the steps of (a) performing the method of claim 31 on apopulation of cells to determine an NPE from at least one cluster ofdatapoints; and (b) determining whether the NPE is within a predefinedrange for the geometric parameter.
 38. The method of claim 37, whereinthe predefined range is within a range of BC and within a range of SDN.39. The method of claim 37, wherein the phenotype is being of adifferent sex.
 40. The method of claim 37, wherein the phenotype isbeing of a different tissue.
 41. The method of claim 37, wherein thephenotype is being of a different species.
 42. The method of claim 37,wherein the phenotype is being of a different state of differentiation.43. The method of claim 42, wherein the geometric parameter is increasedmajor axis.
 44. The method of claim 37, wherein the phenotype is beingat a preselected cell division cycle stage.
 45. The method of claim 44,wherein one stage cycle is S.
 46. The method of claim 45, wherein thegeometric parameter is increased major axis.
 47. The method of claim 44,wherein a stage cycle is selected from the group consisting of G₀, G₁,G₂ and M.
 48. The method of claim 37, wherein the phenotype is being inan apoptotic state.
 49. The method of claim 37, wherein the phenotype isbeing of a disease state.
 50. The method of claim 49, wherein thedisease state is a genetic disease.
 51. The method of claim 50, whereinthe disease state is sickle cell anemia.
 52. The method of claim 50,wherein the disease state is Down's syndrome.
 53. The method of claim49, wherein the disease state is an autoimmune disease.
 54. The methodof claim 37, wherein the phenotype is aneuploidy.
 55. The method ofclaim 37, wherein the phenotype is being neoplastic.
 56. The method ofclaim 55, wherein the geometric parameter is increased major axis. 57.The method of claim 55, wherein the indication of a different cell typeis indicative of a malignant cell.
 58. The method of claim 57, whereinthe geometric parameter is reduced slope of the gradient line.
 59. Themethod of claim 55, wherein the indication of a different cell type isindicative of a metastasizing cell.
 60. The method of claim 59, whereinthe geometric parameter is broadened minor axis.
 61. The method of claim54, wherein the cell is from breast tissue.
 62. The method of claim 54,wherein the cell is from cervical tissue.
 63. The method of claim 54,wherein the cell is from lung tissue.
 64. The method of claim 54,wherein the cell is from tissue selected from the group consisting ofcolon, gastric, lymphatic, intestine and prostate.
 65. The method ofclaim 54, wherein the cell is from tissue selected from the groupconsisting of brain, ovary, testes, bone and exfoliated circulatorytissue.
 66. A device for determining the NPE of a cell, comprising afirst means for measuring a biochemical component of a nucleus from atleast one cell (BC); a second means for measuring the spatialdisplacement (SDN) of the nucleus of at least one cell; and means fordetermining a nuclear packing efficiency (NPE) by correlating the valuesof BC and SDN.